Ubiquitination is a post-translational modification that affects virtually all aspects of cellular function. Despite the importance of ubiquitination, the subset of proteins that are ubiquitinated are largely unknown and it remains difficult to identify proteins that are ubiquitinated in response to specific intracellular signaling pathways. Additionally, although there are over 500 different ubiquitin ligases, there is no straightforward method to identify the specific proteins that are regulated by any of these enzymes. In contrast, analysis of the dynamics of phosphorylation in the proteome has been much more amenable to high-throughput proteomic analysis by the use of mass spectrometry (MS)-based methods. These studies rely on the use of anti-phosphotyrosine antibodies and the selective recovery and enrichment of phosphotyrosine-containing peptides using immunoaffinity purification methods. In this application, we describe the development and use of novel anti-diglycyl-lysine antibodies. This antibody recognizes a unique modification of lysine residues that is seen in ubiquitinated proteins that have been treated with trypsin. Using these antibodies, we have performed the first comprehensive analysis of ubiquitination in cells and tissues. In this application we seek to use this antibody to perform the first analysis of signal dependent ubiquitination as well as the first characterization of the target specificity of an ubiquitin ligase. Our studies focus on netrin-1, a signaling molecule that elicits axonal outgrowth in an ubiquitin-dependent manner, and Smurf1, a ubiquitin ligase implicated in both netrin-1 signaling and neurite outgrowth. Together, the experiments proposed in this application will result in the development of a new and broadly applicable technology that will allow researchers to profile the changes in ubiquitination that occur in a wide variety of cells, signaling pathways, and disease processes. PUBLIC HEALTH RELEVANCE: Ubiquitination is a common cellular mechanism to regulate various aspects of protein function. Ubiquitination is a critical component of synapse plasticity, receptor trafficking, transcription, neuronal morphogenesis, cancer, and numerous brain disorders. Currently, there is no simple and straightforward method to study ubiquitination in cells on a proteome-wide scale. This application describes a new reagent and the use of this reagent in a novel and broadly applicable method to analyze ubiquitination in neurons. Using this method, signaling pathways involved in axonal growth will be investigated.